Compounds isolated from Antrodia cinnamomea and use thereof

ABSTRACT

The present invention relates to novel compounds from  Antrodia cinnamomea  and their use.

FIELD OF THE INVENTION

The present invention is related to a compound from the metabolite ofAntrodin C isolated form Antrodia cinnamomea.

BACKGROUND OF THE INVENTION

Antrodia cinnamomea (Polyporaceae, Aphyllophorales) is well known inTaiwan as a traditional Chinese medicine. It grows only on the innerheartwood wall of the endemic evergreen Cinnamomun kanehirai(Hey)(Lauraceae) in Taiwan. It has been used as treating food to remedytoxication, diarrhea, abdominal pain, hypertension, itchy skin, andliver cancer (Tsai Z T, et al. 1982 Sheng-Yun Publisher Inc.: Taichung,Taiwan, pp 116-117). The compounds of steroid acid (Chen C H, et al.1995 J Nat Prod 58: 1655-1661; Yang S W, et al. 1996 Phytochemistry 41:1389-1392), triterpenoids (Cherng I H, et al, 1995 J Nat Prod 58:365-371; Cherng I W, et al. 1996 Phytochemistry 41: 263-267), diterpenes(Chen C C, et al. 2006 J Nat Prod 69: 689-691), sesquiterpene lactone(Chiang H C, et al. 1995 Phytochemistry, 39, 613-616) and phenyl andbiphenyl (Chiang H C, et al. 1995 Phytochemistry, 39, 613-616; Huang KF, et al. 2001 Chin Pherm J 53: 327-331) were isolated from the fruitingbody of Antrodia cinnamomea, possessing cytotoxic, neuroprotective,anti-inflammatory, apoptotic activities. Moreover, mycelium, anotherpart of Antrodia cinnamomea has antioxidative (Hsiao G., et al. 2003 JAgric Food Chem 51: 3302-3308; Song T Y, et al. 2003 J Agric Food Chem51: 1571-1577), hepatoprotective (Han H F, et al. 2006 Chem Pharm Bull54: 496-500), anti-inflammatory (Shen Y C, et al. 2004 Planta Medica 70:310-314; Hseu Y C, et al. 2005 Int Immunopharmacol 5: 1914-1925),anti-hepatitis B virus (Lee I H, et al. 2002 FEMS Microbiol Lett 209:63-67), vasorelaxation (Wang G. J, et al. 2003 Life Sci 73: 2769-2783)and apoptosis (Song T Y, et al. 2005 J Agric Food Chem 53: 5559-5564)actions.

SUMMARY OF INVENTION

The present invention provide a compound having the formula

wherein R₁ is C₁₋₁₀ carboxylic acid or C₁₋₁₀ ester; R₂ is C₁₋₁₀carboxylic acid or C₁₋₁₀ ester; R₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl; and R₄ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl.

The present invention also provides a composition comprising a compoundhaving the formula

wherein R₁ is C₁₋₁₀ carboxylic acid or C₁₋₁₀ ester; R₂ is C₁₋₁₀carboxylic acid or C₁₋₁₀ ester; R₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl; and R₄ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 HMBC (a) and NOE (b) Correlations of M1.

FIG. 2 HMBC Correlations of M2 and M3.

FIG. 3 TIC of feces (a), bile (b) and plasma (c) sample after oraladministration of Antrodin C at the dose of 50 mg/kg.

FIG. 4 MS spectra (negative mode) of M1-M5 and Antrodin C in the ratfeces, bile and plasma samples.

FIG. 5 The structures of Antrodin C and its metabolites.

FIG. 6 Concentration-time curve of M1 in bile samples after I.V.Antrocin C at the dose of 10 mg/kg (a) and P.O. 50 mg/kg (b).

FIG. 7 The TIC of Bile and Plasma and the UV Spectra of Faeces afterAdministration of Antrodin C in Rats

FIG. 8 The UV spectra of the blank plasma samples and the plasma samplesafter i.v. of M1

DETAIL DESCRIPTION OF THE INVENTION

In this invention, three maleic acid and two succinic acid derivatives(Antrodin A-E) were firstly isolated from the mycelium of Antrodiacinnamomea, and the cytotoxic activity against LLC cells of Antrodin Cand B were confirmed (Nakamura N, et al. 2004 J Nat Prod 7: 46-48).Furthermore, Antrodin C, with the highest amounts in mycelium, exhibitedprotective effect against hepatitis model induced by LPS. Whereas themetabolism study on the compounds of Antrodia cinnamomea were neverreported. In the present invention, the metabolites of Antrodin C in therat bile and feces samples were identified by LC/MS-MS with electrosparyionization (ESI), and the pharmacokinetics of M1 in rat bile wasperformed after oral administration (50 mg/kg) and intravenous injection(10 mg/kg) of Antrodin C by PAD-HPLC.

The present invention provides a compound having the formula

wherein R₁ is C₁₋₁₀ carboxylic acid or C₁₋₁₀ ester; R₂ is C₁₋₁₀carboxylic acid or C₁₋₁₀ ester; R₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl; and R₄ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl.

R₁ or R₂ of the compound is C₁₋₆ carboxylic acid. In the preferredembodiment, R₁ or R₂ is COOH, R₃ is C₁₋₆ alkyl and R₄ is isobutyl. Inthe more preferred embodiment, the compound is(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid,(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 4-methyl ester or(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 1-methyl ester.

The compounds are metabolites of Antrodin C in rats, and the Antrodin Cis isolated from the myvelium Antrodia cinnamomea.

The present invention provides a composition comprising a compoundhaving the formula

wherein R₁ is C₁₋₁₀ carboxylic acid or C₁₋₁₀ ester; R₂ is C₁₋₁₀carboxylic acid or C₁₋₁₀ ester; R₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl; and R₄ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl.

In the preferred embodiment, the compound is(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid,(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 4-methyl ester or(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 1-methyl ester.

The present invention provides the compounds have possessingantioxidation, antimicrobial, antibacterial actions, AChE inhibitoryactivity, antispasmodic or vasorelaxant activities.

The compound of the invention can decrease systolic blood pressure orincrease high density lipoprotein. In addition, the same compound hascentral cholinergic agonism, hepatoprotection, anti-inflammation oranti-tumor activity. Especially, the compound of the invention caninhibit tumor from the cells or tissues selected from the groupconsisting of liver, lung, intestine, bone, blood, lymph and breast. Thesubject accepting the mixture of the invention includes but is notlimited to human, mammal, mouse, rat, horse, pig, chicken, duck, dog andcat.

The present invention also provides a composition, which comprises thecompound of the invention. The composition of the invention can decreasesystolic blood pressure or increase high density lipoprotein. Inaddition, the composition of the invention has central cholinergicagonism, hepatoprotection, anti-inflammation or anti-tumor activity.Especially, the compound of the invention can inhibit tumor from thecells or tissues selected from the group consisting of liver, lung,intestine, bone, blood, lymph and breast. The subject accepting themixture of the invention includes but is not limited to human, mammal,mouse, rat, horse, pig, chicken, duck, dog and cat.

EXAMPLE

Chemicals and Reagents

General anaerobic medium (GAM) broth was purchased from Nissui Co.(Tokyo, Japan). Liquid chromatographic grade solvents, trietylamine,4-dimethylaminopyridine (4-DMAP), silica gel BW-820MH (Fuji Silysia),ODS DM 1020T (Fuji Silysia) for open column chromatography, Merckprecoated Silica gel 60F₂₅₄ (0.25 mm) and Merck RP-18F₂₅₄ (0.25 mm) forTLC analysis were obtained from Wako Pure Chemical Industries Ltd.(Osaka, Japan).

Instruments

Compounds were analyzed by ¹H- and ¹³C-NMR and 2D NMR using a Unity Plus500 (varian) NMR spectrometer with tetramethylsilane as an internalstandard, and chemical shifts are shown as δ values. Intestinal bacteriawere anaerobically incubated using an EAN-140 (Tabai Co., Osaka, Japan).The HPLC instrument was an Agilent 1100 system (Agilent Technologies,Waldbronn, Germany) comprising an Agilent 1100 series binary pump with aphotodiode array detector (PAD) and a series 7725i injector with a 20 μlloop. Data were acquired and integrated using a ChemStation. The HPLCsystem was connected to an Esquire 3000^(plus) mass spectrometer (BrukerDaltonik GmbH, Bremen, Germany) equipped with an ESI source. AllLC/MS-MS data were acquired using Esquire Control software and analyzedusing software from by Bruker Daltonics.

Example 1

Synthesis of M1-M3

Antrodin C (50 mg) was dissolved in 5 ml water, and 1N KOH (0.5 ml) wasadded stirring for 5 min. 1N HCl was used to adjust PH to 8. Afterfiltration, the solution kept under room temperature overnight. Afterfiltration again, the supernatant was lyophilized, and reconstitute bysome MeOH, and then filtered and evaporated in vacuo., yield of M1 was13 mg (26%).

The ¹H and ¹³C-NMR spectra of M1 (Table 1) was very similar to those ofAntrodin C and showed the presence of isobutyl moiety, a3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring, whichsupported by ¹H-¹H COSY

HMQC experiments. But carbonyl carbon (δ 178.9:1), methylene carbon (δ39.7:1′), proton (δ 1.87:1′) and methyne proton (δ 1.56:2′) of isobutylmoiety, benzene carbon conjugated olefine (δ 131.6:1″) and benzeneproton next to that (δ 7.11:2″, 6″) were different from those ofAntrodin C, these all the carbon of M1 were downfield shifted than thoseof Antrodin C and these all the proton of M1 were upfield shifted thanthose of Antrodin C. In the HMBC experiments, long-range correlationswere observed as shown in FIG. 1( a). We decided that Olefine coupling(2-C and 3-C) of M1 is Z because NOE was observed between 1′-H and3′4′-H or 6″-H in the NOESY spectrum of M1 (FIG. 1( b)). According tothese results, M1 was defined as(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]-phenyl}but-2-enedioicacid. Anhydride M4 (Antrodin A) and dicarboxylic acid M1 were convertedeach other by acid and base condition.

Antrodin A (500 mg) was dissolved in 1 ml MeOH, and trietylamine (0.2ml, 1.6 mmol) and 4-dimethylaminopyridine (4-DMAP, 13.4 mg, 0.11 mmol)were added to the solution stirring for 20 h at 25° C. And then themixture was chromatographed by a open ODS column eluting with methanoland water (30:70→100:0), the fraction containing M2 and M3 wereevaporated in vacuo, and then analyzed by NMR and LC/MS. The data of¹H-NMR and ¹³C-NMR of M1-M3 were showed in Table 1, 2. The ¹H and¹³C-NMR spectra of M2 and M3 were also similar to those of M1 except formethoxy groups and showed the presence of isobutyl moiety, a3-methyl-2-butenyloxy moiety, and a para-substituted benzene ring. Inthe HMBC experiments, methylene proton of isobutyl moiety (δ 2.16:1′)and carbonyl carbon (δ 174.0:1) of M2 showed long-range correlation, andmethylene proton of isobutyl moiety (δ 2.14:1′) and carbonyl carbon (δ171.4:1) of M3 showed long-range correlation (FIG. 2). The structure ofM2 and M3 were defined as(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 4-ester and(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 1-methyl ester, respectively.

TABLE 1 ¹H-NMR Spectral Data of M1 (D₂O), M2 and M3 (CD₃OD) (δ ppm, J =Hz) M1 M2 M3 1′ 1.87 (2H, d, J = 6.5) 2.16 (2H, d, J = 7.0) 2.14 (2H, d,J = 7.0) 2′ 1.56 (1H, m) 1.69 (1H, m) 1.69 (1H, m) 3′, 4′ 0.72 (6H, d, J= 6.5) 0.81 (6H, d, J = 7.0) 0.80 (6H, d, J = 6.5) 2″, 6″ 7.11 (2H, d, J= 8.5) 7.14 (2H, d, J = 9.0) 7.20 (2H, d, J = 9.0) 3″, 5″ 6.88 (2H, d, J= 8.5) 6.91 (2H, d, J = 9.0) 6.91 (2H, d, J = 9.0) 1″′ 4.50 (2H, d, J =6.5) 4.54 (2H, d, J = 6.5) 4.54 (2H, d, J = 6.5) 2″′ 5.41 (1H, brs) 5.46(1H, m) 5.46 (1H, m) 4″′ 1.68 (3H, s) 1.77 (3H, s) 1.77 (3H, s) 5″′ 1.64(3H, s) 1.75 (3H, s) 1.75 (3H, s) —OMe — 3.82 (3H, s) 3.72 (3H, s)

TABLE 2 ¹³C-NMR Spectral Data of M1 (D₂O), M2 and M3 (CD₃OD) (δ ppm) M1M2 M3 1 178.9 174.0* 171.4* 2 140.3 144.0 134.3 3 137.5 136.3 145.4 4166.1 * * 1′ 39.7 40.0 40.0 2′ 27.2 29.0 29.0 3′, 4′ 22.2 22.8 22.8 1″131.6 128.5 128.5 2″, 6″ 130.5 131.4 131.0 3″, 5″ 114.8 115.5 115.5 4″157.0 160.0 160.0 1″′ 65.3 65.9 65.9 2″′ 118.4 121.1 121.1 3″′ 141.6138.7 138.7 4″′ 25.1 25.9 25.9 5″′ 17.3 18.2 18.2 —OMe — ** **

Example 2

Treatment of Animals

Male Wistar rats (9 weeks old) purchased from SLC Co. (Hamamastu,Japan), were fed with standard laboratory chow for one week, fastedovernight and given free access to water before drug administration.Urine and feces samples were collected while the rats remained isolatedin metabolic cages. The animals were light anesthetized with diethylether during surgical procedures. Bile samples (n=5) was collected bycannulating a polyethylene tube (PE-10) into the rat bile duct atintervals of 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h after oral(50 mg/kg) and intravenous (10 mg/kg) administration of Antrodin C. Theblood sample was collected from the inferior vena cava using aheparinized injector when the abdomen was exposed by a midline abdominalincision after administration. The blood samples were centrifuged at8000×g for 15 min to separate the plasma, and then all samples werestored at −20° C. for later analysis.

Sample Preparation for Analysis

Thawed urine and bile samples (0.5 ml) dissolved in 3 volumes ofacetonitrile, and then centrifuged at 8000×g for 15 min. The supernatantwas passed through a 0.45 μm Millipore syringe filter (Nihon Millipore,Tokyo, Japan) for LC/MS-MS analysis. Plasma samples were passed throughSolid Phase Extraction cartridges (Waters Co., Milford, U.S.A.) that hadbeen washed with 3 ml of acetonitrile and equilibrated with 6 ml ofwater. The constituents were eluted with 2-3 ml of acetonitrile from thecartridge, then the eluate was evaporated under a stream of nitrogen at35° C. to leave a residue that was dissolved in 100 μl of acetonitrilefor LC/MS-MS analysis. The bile samples for pharmacokinetic study, whichcontaining M2, M3 and M4 were diluted by same volume water, and thenincubated in 37° C. bath for 12 h, the M2-M4 would thoroughly convertedto M1. After treating the bile sample as described above, the amount ofM1 was calculated by PAD-HPLC.

Identification of Metabolites in Rat feces, Bile and Plasma

The metabolites in feces, bile and plasma were analyzed by LC/MS-MS. TheLC/MS-MS equipment comprised a column containing TSK gel ODS-80 Ts(particle size, 5 μm; 4.6×150 mm i.d., Tosoh Co., Tokyo, Japan). Sampleswere eluted through the column with 0.1% AcOH and acetonitrile (35:65)at a flow rate of 1 ml/min at 30° C. The standard negative ion mode wasselected under the following conditions: full scan range, 50-800 m/z;scan resolution, 13000 m/z/sec; nebulizer, 50.0 psi; dry gas, 10.0l/min; dry temperature, 360° C. Full scanning in the region of m/z 50 to600 assigned several peaks to Antrodin C and the metabolites in the TLCwhen compared with those of blank samples (FIG. 3). The MS spectrarevealed intense ion peaks at m/z 331, 345, 345, 313, 312 and 328 [M-H]⁻as M1, M2, M3, M4, M5 and Antrodin C, respectively (FIG. 4, Table 3). Inthe feces, metabolites were M1-M3, M5 with original Antrodin C; in thebile were M2-4; and in the plasma were M1 with another unknown peak.There was neither metabolite nor Antrodin C in the urine sample.Comparing to standard materials and synthesized compounds, the peak inthe MS profile at m/z 328 [M-H]⁻ with retention time (t_(R))=8.2 min wasderived from Antrodin C (MW 329), and M1 (m/z 331 [M-H]⁻, t_(R)=4.4 min)was dicarboxylic acid by hydrolysis of Antrodin C; M2 and M3 (m/z 345[M-H]⁻, t_(R)=7.7 and 8.4 min), which were 14 larger than those of M1,were two kinds of monomethyl esters of M1; M4 (m/z 313 [M-H]⁻,t_(R)=21.6 min) was Antrodin A and M5 (m/z 312 [M-H]⁻, t_(R)=11.5 min)was Antrodin B. The structures of Antrodin C and its metabolites wereshown in FIG. 5.

TABLE 3 Retention time (t_(R)) and MS spectra of Antrodin C and itsmetabolites t_(R) MS spectra (Negative mode) Antrodin C 8.2 328, 259,242, 216 M1 4.4 331, 287, 218, 132 M2, M3 7.7, 8.4 345, 313, 244, 232,189 M4 21.6 313, 244 M5 11.5 312, 243, 200 Unknown metabolite 10.8 319,301, 257, 179, 163, 135Metabolism of Antrodin C and Metabolites by Intestinal Bacteria In Vitro

Mixtures of rat (RIB) or human (HIB) intestinal bacteria (5 g each)prepared as described (Xie L H, et al. 2003 Biol Pharm Bull 51:378-384), together with Antrodin C (5 mg) dissolved in Tween 20 (0.5ml), M1 (5 mg) dissolved in water (1.0 ml) or rat bile samples (10 ml)with metabolites M2-M4, which were collected after oral administrationof Antrodin C, were added to GAM broth (50 ml), and anaerobicallyincubated at 37° C. for 3 d. The incubation mixture was extracted with 3volumes of acetonitrile, and then passed through a 0.45 μm filter. Then,Antrodin C was converted to M5 (Antrodin B). Moreover, the metabolites(M2-M4) in bile samples, which were collected after oral administrationof Antrodin C in rats, could absolutely transferred to M1 after 30 minincubation. Whereas M1 was not metabolized by intestinal bacteria flora,although prolonged the incubation time to 3 d.

Validation of M1 by PAD-HPLC

Linearity: M1 was dissolved in rat blank bile to prepare seven dilutionsof standard solutions. Response linearity was determined for the sevenconcentrations after three injections for each level. The limit ofdetection (LOD) of the method for each constituent was established whenthe signal to noise ratio (S/N) was 5.

Accuracy: Intra- and inter-assay variability was determined by analyzinghigh, medium and low standard concentrations of rat bile five times onthe same day and continuously for 5 d, respectively.

Recovery: Two standard concentrations were mixed with rat bile samplesafter the oral administration of Antrodin C with a known amount of M1,and recovery rates of the added amounts were calculated.

Stability: Three concentrations of bile samples that had been preparedfor PAD-HPLC analysis were placed at room temperature for 12 h, or in arefrigerator at 4° C. for 1, 3 and 5 d. The average peak areas ofconstituents in the samples and relative standard deviation (RSD) werecalculated.

Validation of PAD-HPLC Quantitation

The regression equation of M1 in rat bile sample was Y=610.22X−3.94;γ=0.9998; and the linearity range was 0.05-2.0 μg/ml. Intra-day andinter-day (n=5) variations of M1 in rat bile samples were shown in Table4. The CV did not exceed 6%, and the accuracy rates were all within85-110%. CV values of recovery rates were shown in Table 5, which wereless than 10% at low and high concentrations with recovery rates of 93.4and 99.6%. The stability test showed that relative standard deviationremained within 5% under all the conditions; therefore, the samples werestable during the test. Thus, the accuracy, recovery, and stabilitytests met the criteria for quantitative determinations in bile samples.

TABLE 4 Intraday and Interday (n = 5) Variations of M1 in Rat Bile Added(μg) Found (μg) Accuracy (%) CV (%) Intraday 0.05 0.0456 ± 0.0018 91.23.9 0.5 0.533 ± 0.008 106.6 1.5 2.0 1.99 ± 0.04 99.5 2.0 Interday 0.050.0438 ± 0.0026 87.6 5.9 0.5 0.443 ± 0.009 88.6 2.0 2.0 1.98 ± 0.06 99.03.0

TABLE 5 Recovery of M1 in Rat Bile Added (μg) Found (μg) Recovery (%) CV(%) 0.05 0.0467 ± 0.0045 93.4 9.6 0.5 0.498 ± 0.020 99.6 4.0Pharmacokinetics of M1 in Rat Bile

The concentration-time data in rat bile (n=5) were computer fitted usinga program of Pharmacokineitics 3p97 edited by the MathematicsPharmacological Committee, Chinese Pharmacological Society. Thefollowing pharmacokinetic parameters were obtained: half-time ofabsorption phase (t_(1/2 (Kα))) and half-time of elimination phase(t_(1/2 (Kβ))) in the bile samples after oral administration of AntrodinC at the dose of 50 mg/kg. The area under the concentration-time curve(AUC_((i.v.)) and AUC_((p.o.))) was calculated by the statistical momentmethod of non-compartmental pharmacokinetic analysis. And then clearance(Cl_(m, b)) and absolute bioavailability (F_(m, b)) were calculated bythe equations as following: Cl_(m, b)(ml/h·kg)=Dose_((i.v.))/AUC_((i.v.)) and F_(m, b)(%)=AUC_((p.o.))·Dose_((i.v.))/[AUC_((i.v.))·Dose_((p.o.))]. Data wereexpressed mean and standard deviation (S.D.) for each group.

The concentrations of M1 in bile samples were calculated after P.O.administration of 50 mg/kg and I.V. 10 mg/kg of Antrodin C. Theconcentration-time curves of M1 were shown in FIG. 6. Thepharmacokinetic parameters were shown in Table 6. After oraladministration, t_(1/2 (kα)) and t_(1/2 (kβ)) were 0.95 h and 12.64 h,respectively. AUC_(0-lim) were 1.61 (P.O.) and 1.68 h mg/ml (I.V.),Cl_(m.b). was 5.96 ml/h·kg and F_(m.b.) was 19.43(%). Accumulatedexcretion ratio of Antrodin C were 5.46±1.62% (P.O.) and 56.85±13.40(I.V.). Therefore, Antrodin C was very quickly not only absorbed fromgastrointestine, but also metabolized in the liver. The mainly excretionwas bile-feces pathway in rats.

TABLE 6 Pharmacokinetic parameters of M1 in rat bile samples after P.O.and I.V. of Antrodin C I.V. (10 mg/kg) P.O. (50 mg/kg) Cl_(m.b.)t_(1/2 (kα)) t_(1/2 (kβ)) AUC_(0-lim) AUC_(0-lim) (ml/ F_(m.b.) (h) (h)(h mg/ml) (h mg/ml) h kg) (%) 0.95 ± 0.07 12.64 ± 2.24 1.61 ± 0.58 1.68± 0.31 5.96 19.43

Example 3

Repeat example 2, results are shown in FIG. 7 and FIG. 8

1. An isolated compound having the formula

wherein R₁ is C₁₋₁₀ carboxylic acid or C₁₋₁₀ ester; R₂ is C₁₋₁₀carboxylic acid or C₁₋₁₀ ester; R₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl; and R₄ is H, C₂₋₁₀ alkyl, C₂₋₁₀ alkenyl or, C2-10alkynyl.
 2. The isolated compound of claim 1, wherein R₁ or R₂ is C₁₋₆carboxylic acid.
 3. The isolated compound of claim 1, wherein R₁ or R₂is COOH.
 4. The isolated compound of claim 1, wherein R₃ is C₁₋₆ alkyl.5. The isolated compound of claim 1, wherein R₄ is isobutyl.
 6. Theisolated compound of claim 1, which is(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid,(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 4-methyl ester or(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 1-methyl easter.
 7. The isolated compound of claim 6, wherein thecompound is metabolites of Antrodin C in rats.
 8. A compositioncomprising an isolated compound having the formula

wherein R₁ is C₁₋₁₀ carboxylic acid or C₁₋₁₀ ester; R₂ is C₁₋₁₀carboxylic acid or C₁₋₁₀ ester; R₃ is H, C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl orC₂₋₁₀ alkynyl; and R₄ is H, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl or isobutyl. 9.The composition of claim 8, wherein the isolated compound is(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid,(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 4-methyl ester or(2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 1-methyl easter.